(a) Field of the Invention
The present invention relates to a novel use of a transfected cell as a biosensor, wherein the transfected cell expresses a receptor specific to a bio-signal transmitter, and a highly sensitive detector and a highly sensitive detecting method of a bio-signal transmitter using the biosensor.
(b) Description of the Related Art
Bio-signal transmitters generally refer to a series of materials including all kinds of hormones, neurotransmitters, and other intercellular signal transmitters that are released from many kinds of tissues or cells in the body and transmit information to adjacent tissues or cells. Of them, the neurotransmitters may be generally classified into four classes, i.e., an amino acid-based class (e.g., acetylcholine, glycine, aspartic acid, glutamate, and the like), an amine-based class (e.g., dopamine, adrenalin (epinephrine), noradrenalin, Gamma-aminobutyric acid (GABA), and the like), a peptide-based class (e.g., vasopressin, and the like.), and a fatty acid-based class (e.g., histamine, serotonin, and the like). It has been known that the above compounds are released into synapses and participate in transmitting information between neurons. Such neurotransmitter release plays an important role in neurotransmission between neurons, and a very small change of the released amount affects the neurotransmission. Nevertheless, since the released amount is extremely small, it has been difficult as well as important to achieve highly sensitive detection and quantification of the bio-signal transmitter release.
Although there has been much research into the development of techniques of detecting and measuring the bio-signal transmitter release from bio-samples, none of the research has been successful in developing techniques that simultaneously satisfy high sensitivity together with a higher time and spatial resolution required for quantifying the release from a single cell level. Up to the present, most of the developed techniques have technical limits in directly detecting the bio-signal transmitter release from each position of the neural membrane.
Of the developed techniques, electrochemical methods, such as amperometry and fast cyclic voltametry, are useful for fast and highly sensitive detection of a bio-signal transmitter at an isolated position where the released bio-signal transmitter is readily oxidized or reduced. However, these methods are limited in the number of bio-signal transmitters to be detected. In order to solve this problem, there have been several attempts to improve the number of bio-signal transmitters to be detected by enzymatically converting the bio-signal transmitters into easily oxidizable intermediates. However, the enzymatic conversion step is too slow to detect the release from a single cell level.
Recently, a sniffer-patch detecting technique has been developed as an alternative to the above techniques. The sniffer-patch method, which was firstly used for detecting a spontaneous release of Ach (acetylcholine) from a developing growth cone, can provide a highly sensitive biological detection system utilizing a high affinity of a ligand-gated ion channel to a natural bio-signal transmitter. This method is based on a concept of excising a small patch of membrane on which many receptors exist from a donor cell using an outside-out configuration of a patch-clamp recording method, and detecting the release position of the bio-signal transmitter using the excised patch. The first step of the detection is to excise a membrane patch from a donor cell. Thereafter, the excised patch is voltage-clamped to a potential in a range capable of providing an impulse sufficient for ion transfer through the channel to be detected while preventing activation of voltage-gated ion channels.
Since the sniffer-patch method uses a membrane of a proper donor cell as a biosensor, it is required to develop donor cells that are suitable for a biosensor in order to achieve higher detection efficiency.